Search

Semiconductor nanocrystals (quantum dots - QDs) possess unique photophysical properties that make them highly interesting for many biochemical applications. Besides their common use as fluorophores in conventional spectroscopy and microscopy, QDs are well-suited for studying Forster resonance energy transfer (FRET). Size-dependent broadband absorption and narrow emission bands offer several advantages for the use of QDs both as FRET donors and acceptors. QD-based FRET pairs can be efficiently used as biological and chemical sensors for highly sensitive multiplexed detection. In this contribution we present the use of several commercially available QDs (Qdot® Nanocrystals - Invitrogen) as FRET donors in combination with commercial organic dyes as FRET acceptors. In order to investigate the FRET process within our donor-acceptor pairs, we used biotinylated QDs and streptavidin-labeled dyes. The well-known biotinstreptavidin molecular recognition enables effective FRET from QDs to dye molecules and provides defined distances between donor and acceptor. Steady-state and time-resolved fluorescence measurements were performed in order to investigate QD-to-dye FRET. Despite a thick polymer shell around the QDs, our results demonstrate the potential of these QDs as efficient donors both for steady-state and time-resolved FRET applications in nano-biotechnology.

Semiconductor quantum dots (QDs) are highly interesting fluorophores for a large variety of spectroscopic applications. Although their fluorescence properties are well investigated, accurate size determination of QDs is still a problem. TEM techniques can image the inorganic core/shell system of QDs, but size determination of polymer coated QDs is difficult. SEC (size exclusion chromatography) compares the QD size only with standard polymers and their sizes, and is therefore not easy to use on nanoparticles. As QDs are fluorescent, single molecule spectroscopy methods such as fluorescence correlation spectroscopy (FCS) can be used to determine QDs diffusion coefficients and hence their hydrodynamic radii. Moreover, this method for size determination requires only very low QD concentrations, which is a mayor advantage compared to other techniques such as dynamic light scattering. Within our contribution we present the size determination of commercially available and self-modified QDs with FCS. The commercial QDs (QD525, QD565, QD605, QD655 and QD705 - purchased from Invitrogen Inc.) have a rather thick polymer shell and are functionalized with streptavidin, biotin or carboxylic groups. The self-modified QDs consist of the same commercial core/shell QDs and are modified with a polymer shell and several bio-functionalization groups. For all nanoparticles the diffusion coefficients were measured by FCS and the hydrodynamic radii were calculated according to the Stokes-Einstein equation. The obtained results are in good agreement with the size information provided by Invitrogen Inc., which demonstrates that FCS is an important technique for QD size determination at very low concentrations.

Holographic surface relief gratings written in azobenzene containing films were studied for the use as masters for polymeric thin film distributed feedback (DFB) lasers. Light induced mass transport driven by E-Z isomerization in azobenzene containing materials have shown to be attractive for all optical and one-step fabrication of periodic surface structures with varying parameters for different optical applications. Based on new azobenzene materials and their holographic processing deep surface relief gratings were generated with grating pitches in the range of 400 nm as resonant structures for second order DFB lasers emitting in the VIS range. Nanoimprint techniques enabled multiple duplications of azobenzene master gratings in UV adhesives. The replicas were coated via spin casting with thin films of red light emitting polymer layers to form DFB thin film lasers. These active layers are guest-host-systems consisting of an UV-light absorbing conjugated polymer as host transferring its excitation via Forster resonant energy transfer to a red emitting conjugated polymer. Simple adjustment of grating depth via controlling of illumination time allowed it to investigate the influence of the corrugation depth and thereby the coupling of laser light and grating on the lasing behavior of second order DFB lasers in the red region. For this purpose multiple surface structures with different corrugation depths of up to 130 nm were generated holographically, duplicated and coated.

An innovative approach for voltage-tunable optical gratings based on dielectric elastomer actuators (DEAs) using electro active polymers is presented. Sinusoidal surface gratings, holographically written into azobenzene containing films, are transferred via nanoimprinting to DEAs of different carrier materials. We demonstrate that the surface relief deformation depends on the mechanical and geometrical properties of the actuators. The tested DEAs were made using commercially available elastomers, including a tri-block copolymer poly-styrene-ethylene-butadiene-styrene (SEBS), a silicone polydimethylsiloxane rubber (PDMS) and commonly used polyacrylic glue. The polyacrylic glue is ready to use, whereas the SEBS and the PDMS precursors have to be processed into thin films via different casting methods. The DEA material was pre-stretched, fixed to a stiff frame and coated with stretchable electrodes in appropriate designs. Since the actuation strain of the DEA depends strongly upon the conditions such as material properties, pre-stretch and geometry, the desired voltage-controllable deformations can be optimized during manufacturing of the DEA and also in the choice of materials in the grating transfer process. A full characterization of the grating deformation includes measurements of the grating pitch and depth modulation, plus the change of the diffraction angle and efficiency. The structural surface distortion was characterized by measuring the shape of the transmitted and diffracted laser beam with a beam profiling system while applying an electro-mechanical stress to the grating. Such surface distortions may lead to decreasing diffraction efficiency and lower beam quality. With properly chosen manufacturing parameters, we found a period shift of up to 9 % in a grating with 1 μm pitch. To describe the optical behavior, a model based on independently measured material parameters is presented.

An optical multiplexed homogeneous (liquid phase) immunoassay based on FRET from a terbium complex to eight different fluorescent dyes is presented. We achieved highly sensitive parallel detection of four different lung cancer specific tumor markers (CEA, NSE, SCC and CYFRA21-1) within a single assay and show a proof-of-principle for 5- fold multiplexing. The method is well suited for fast and low-cost miniaturized point-of-care testing as well as for highthroughput screening in a broad range of in-vitro diagnostic applications.

Homogeneous immunoassays have the benefit that they do not require any time-consuming separation steps. FRET is one of the most sensitive homogeneous methods used for immunoassays. Due to their extremely strong absorption over a broad wavelength range the use of quantum dots as FRET acceptors allows for large Forster radii, an important advantage for assays in the 5 to 10 nm distance range. Moreover, because of their size-tunable emission, quantum dots of different sizes can be used with a single donor for the detection of different analytes (multiplexing). As the use of organic dyes with short fluorescence decay times as donors is known to be inefficient with quantum dot acceptors, lanthanide complexes with long luminescence decays are very efficient alternatives. In this contribution we present the application of commercially available biocompatible CdSe/ZnS core/shell quantum dots as multiplexing FRET acceptors together with a single terbium complex as donor in a homogeneous immunoassay system. Forster radii of 10 nm and FRET efficiencies of 75 % are demonstrated. The high sensitivity of the terbium-to-quantum dot FRET assay is shown by sub-100-femtomolar detection limits for two different quantum dots (emitting at 605 and 655 nm) within the same biotin-streptavidin assay. Direct comparison to the FRET immunoassay “gold standard” (FRET from Eu-TBP to APC) yields a three orders of magnitude sensitivity improvement, demonstrating the big advantages of quantum dots not only for multiplexing but also for highly sensitive nanoscale analysis. Keywords: Quantum Dots, Terbium, Time-Resolved Luminescence, FRET, Multiplexing

Quantum dots (QDs) are common as luminescing markers for imaging in biological applications because their optical properties seem to be inert against their surrounding solvent. This, together with broad and strong absorption bands and intense, sharp tuneable luminescence bands, makes them interesting candidates for methods utilizing Forster Resonance Energy Transfer (FRET), e. g. for sensitive homogeneous fluoroimmunoassays (FIA). In this work we demonstrate energy transfer from Eu3+-trisbipyridin (Eu-TBP) donors to CdSe-ZnS-QD acceptors in solutions with and without serum. The QDs are commercially available CdSe-ZnS core-shell particles emitting at 655 nm (QD655). The FRET system was achieved by the binding of the streptavidin conjugated donors with the biotin conjugated acceptors. After excitation of Eu-TBP and as result of the energy transfer, the luminescence of the QD655 acceptors also showed lengthened decay times like the donors. The energy transfer efficiency, as calculated from the decay times of the bound and the unbound components, amounted to 37%. The Forster-radius, estimated from the absorption and emission bands, was ca. 77Å. The effective binding ratio, which not only depends on the ratio of binding pairs but also on unspecific binding, was obtained from the donor emission dependent on the concentration. As serum promotes unspecific binding, the overall FRET efficiency of the assay was reduced. We conclude that QDs are good substitutes for acceptors in FRET if combined with slow decay donors like Europium. The investigation of the influence of the serum provides guidance towards improving binding properties of QD assays.© (2006) COPYRIGHT SPIE--The International Society for Optical Engineering. Downloading of the abstract is permitted for personal use only.

Due to their extraordinary photophysical properties CdSe/ZnS core/shell nanocrystals (quantum dots) are excellent luminescence dyes for fluorescence resonance energy transfer (FRET) systems. By using a supramolecular lanthanide complex with central terbium cation as energy donor, we show that commercially available biocompatible biotinilated quantum dots are excellent energy acceptors in a time-resolved FRET fluoroimmunoassay (FRET-FIA) using streptavidin-biotin binding as biological recognition process. The efficient energy transfer is demonstrated by quantum dot emission sensitization and a thousandfold increase of the nanocrystal luminescence decay time. A Foerster Radius of 90 a and a picomolar detection limit were achieved in quantum dot borate buffer. Regarding biological applications the influence of bovine serum albumin (BSA) and sodium azide (a frequently used preservative) to the luminescence behaviour of our FRET-system is reported.

Two examples of our biophotonic research utilizing nanoparticles are presented, namely laser-based fluoroimmuno analysis and in-vivo optical oxygen monitoring. Results of the work include significantly enhanced sensitivity of a homogeneous fluorescence immunoassay and markedly improved spatial resolution of oxygen gradients in root nodules of a legume species.